The uridine-rich small nuclear RNAs (U-snRNAs) play an integral role in the expression of virtually all other genes. Included in this list are intron-containing polyadenylated messenger RNAs, histone mRNAs, and ribosomal RNA. It is quite surprising then how little is known about the biosynthesis of these noncoding RNAs. These RNA polymerase II transcripts undergo a single endonucleolytic cleavage event, which accounts for their entire nuclear RNA processing. A poorly understood complex of proteins, termed the Integrator complex, carries out this reaction through a mechanism that is currently unknown. We have a designed a fluorescence-based reporter system that results in robust GFP expression in the event that a component of this 3'end processing machinery is debilitated. This reporter has been used to successfully carry out a genome-wide RNAi screen and has resulted in the identification of novel components of this machinery. The goals of this research plan are to optimize and develop this reporter assay for the high-throughput screening of a compound library. This is further broken down into two Specific Aims: 1) To develop the assay in a miniaturized form either in 96 well or 384-well format and to optimize the assay to maximize signal-to-noise ratio, variability, and achieve a Z'score consistent with an excellent assay. 2) To configure the assay for high-throughput screening, which would include developing counterscreens, secondary screens, and to perform a pilot screen with a small collection of diverse compounds. Successful completion of this research plan will hopefully result in acceptance to use the NIH MLPCN, which may result in the identification of novel compounds that inhibit the 3'end formation of snRNA. These novel compounds will be used to interrogate the complex of factors involved in this process, which will lead to further mechanistic insight into this poorly understood biological process. PUBLIC HEALTH RELEVANCE: The biosynthesis of small nuclear RNA (snRNA) is a fundamental event that must occur for proper gene expression. The inappropriate accumulation of functional snRNA can lead to human disease. By increasing our understanding of this basic biological process we can begin to formulate strategies to address these diseases.